Microcrystalline Y Receptor Agonists

ABSTRACT

The disclosure provides microcrystals of Y receptor agonists; microcrystalline pellets of Y receptor agonists, and microcrystalline suspensions of Y receptor agonists. Pharmaceutical compositions containing these microcrystals, microcrystalline pellets, and microcrystalline suspensions have prolonged pharmacokinetic profiles making them useful for once daily or once weekly administration.

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/363,200filed Jul. 9, 2010, the disclosure of which is incorporated herein.

FIELD

The disclosure provides compounds, compositions and pharmaceuticalformulations of microcrystalline Y receptor agonists.

SEQUENCE LISTING

A computer-readable copy of a sequence listing in text format (.txt) issubmitted as part of the specification.

BACKGROUND

Protein crystals have shown significant benefits in the delivery ofbioactives to achieve high concentration, low viscosity, extendedrelease profiles, and improved stability. Li et al, Acta Cryst.,F63:599-601 (2007); Basu et al, Expert Opin. Biol. Ther., 4(3):301-317(2004); Yang et al, PNAS, 100(12):6934-6939 (2003). Some proteins andpeptides have been crystallized alone or co-crystallized with zinc in anattempt to make extended release products. Y receptor agonists, complexcompounds that often possess inadequate stability and short half-life,have limited delivery options. There is a need in the art forformulations of Y receptor agonists that have extended releaseproperties and that have suitable stability. The disclosure is directedto these, as well as other, important ends.

SUMMARY

The disclosure is based on the unexpected discovered that microcrystalsof Y receptor agonists may be produced that have extendedpharmacokinetic profiles. Therefore, pharmaceutical compositionscontaining the Y receptor agonists may be administered to the patientonce per day, once every other day, or once per week.

The disclosure provides microcrystals of Y receptor agonists, whereinthe microcrystals have a particle size from about 1 micron to about 25microns and are birefringent under a polarized light microscope. In oneembodiment, the microcrystals are complexed with metals. In oneembodiment, the microcrystals are coated with polyamino. In oneembodiment, the microcrystals are complexed with metals and coated withpolyamino acids.

The disclosure provides methods for preparing compositions comprisingmicrocrystals of Y receptor agonists by (i) dissolving a Y receptoragonist peptide in an aqueous solution at a pH of about 3 to about 5,wherein the aqueous solution optionally contains a dissolution agent tofacilitate dissolution of the Y receptor agonist, to produce a firstaqueous solution having the Y receptor agonist peptide dissolvedtherein; (ii) adjusting the pH of the first aqueous solution upward to apH of about 4.0 to about 7.5, to produce a second aqueous solutionhaving the Y receptor agonist peptide dissolved therein; and (iii)stirring the second aqueous solution at a temperature from about 1° C.to about 10° C. for more than one day; thereby producing thecomposition. The compositions may be referred to as microcrystallinesuspensions. Following the additional step of drying, the compositionsmay be referred to as microcrystalline pellets. In one embodiment, themethods may further comprise the addition of metals to form metalcomplexes of the microcrystals. In one embodiment, the methods mayfurther comprise the addition of polyamino acids to form microcrystalscoated with polyamino acids. In one embodiment, the methods may furthercomprise the addition of metals and polyamino acids to formmetal-complexed microcrystals coated with polyamino acids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a microscopic picture of microcrystals of the Y receptoragonist peptide having SEQ ID NO:438.

FIG. 2 is a polarized light microscopic picture showing that themicrocrystals of the Y receptor agonist peptide having SEQ ID NO:438exhibit birefringence.

FIG. 3 is a microscopic picture of microcrystals of the Y receptoragonist peptide having SEQ ID NO:281.

FIG. 4 is a microscopic picture of microcrystals of the Y receptoragonist peptide having SEQ ID NO:439.

FIG. 5 is a microscopic picture of microcrystalline pellets of the Yreceptor agonist peptide having SEQ ID NO:438.

FIG. 6 shows the pK profile (semi-log profile scale) of the followingformulations administered to rats: Group 2: a solution containing thepeptide of SEQ ID NO:438; Group 3: a microcrystalline suspensioncontaining the peptide of SEQ ID NO:438; Group 4: a zinc-complexedmicrocrystalline suspension of the peptide of SEQ ID NO:438, where theratio of peptide:zinc was 3:1; and Group 5: a zinc-complexedmicrocrystalline suspension of the peptide of SEQ ID NO:438, where theratio of peptide:zinc was 1:1.

FIG. 7 shows the body weight loss in rats after a single subcutaneousinjection (5 mg/kg) of the following formulations: Group 1: vehicle;Group 2: a solution containing the peptide of SEQ ID NO:438; Group 3: amicrocrystalline suspension containing the peptide of SEQ ID NO:438;Group 4: a zinc-complexed microcrystalline suspension of the peptide ofSEQ ID NO:438, where the ratio of peptide:zinc was 3:1; and Group 5: azinc-complexed microcrystalline suspension of the peptide of SEQ IDNO:438, where the ratio of peptide:zinc was 1:1.

FIG. 8 shows the cumulative food intake on female NH/Swiss mice after asingle subcutaneous injection (100 μg/kg) of: (i) vehicle; (ii) asolution containing the peptide of SEQ ID NO:438; (iii) amicrocrystalline suspension of the peptide of SEQ ID NO:438; (iv) apoly-L-glutamic acid-coated microcrystalline suspension of peptide ofSEQ ID NO:438; and (v) a peptide of SEQ ID NO:438 modified withpolyethylene glycol.

FIG. 9 shows the change from base line of food intake on rats (n=8)following a subcutaneous injection (1 mg/kg) of (i) vehicle; (ii) asolution containing the peptide of SEQ ID NO:438; (iii) amicrocrystalline suspension of the peptide of SEQ ID NO:438; (iv) amicrocrystalline suspension of peptide of SEQ ID NO:438 coated withpoly-L-glutamic acid.

DETAILED DESCRIPTION

The disclosure is based on the surprising and advantageous benefits ofmicrocrystals of Y receptor agonist peptides. It has been discoveredthat microcrystals of Y receptor agonist peptides have improvements withregard to, e.g., increased efficacy, extended pharmacokinetic profileand bioavailability (e.g., allowing for once daily or once weeklyadministration), and increased stability, relative to that observed withnoncrystalline solutions of Y receptor agonist peptides. This results inimproved efficacy with regard to, e.g., patient compliance, reductionsin body weight, weight loss, reduction in food intake, and beneficialalteration in body composition (e.g., fat-specific weight loss, and/orlean-sparing weight loss), relative to the efficacy observed withnoncrystalline solutions of such Y receptor agonists.

The term “microcrystals” refers to Y receptor agonist peptides having aparticle size ranging from about 1 micron to about 25 microns. In oneembodiment, “microcrystals” refers to Y receptor agonist peptides havinga particle size ranging from about 1 micron to about 25 microns, wherethe microcrystals are birefringent (i.e., have two indices ofrefraction) under polarized light. In one embodiment, “microcrystals”refers to Y receptor agonist peptides having a particle size rangingfrom about 1 micron to about 25 microns, where the microcrystals arebirefringent (i.e., have two indices of refraction) under polarizedlight, and where the microcrystals have an alpha-helical structure, abeta-sheet secondary structure, or an alpha-helical structure and abeta-sheet secondary structure when viewed by infrared spectroscopy(e.g., using an FTIR spectrometer). In one embodiment, the microcrystalshave a particle size ranging from about 1 micron to about 15 microns. Inone embodiment, the microcrystals have a particle size ranging fromabout 1 micron to about 10 microns. In one embodiment, the microcrystalshave a particle size ranging from about 5 microns to about 15 microns.In one embodiment, the microcrystals have a particle size ranging fromabout 5 microns to about 10 microns. In another embodiment, themicrocrystals have (i) oval-shaped crystals; (ii) blade-like crystals;or (iii) oval-shaped crystals and blade-like crystals. Microcrystals areshown in FIGS. 1-5. FIG. 2 shows that the microcrystals are birefringentwhen viewed under a polarized light microscope. Microcrystals having anoval shape are shown in FIGS. 1 and 3. Microcrystals having a thin bladeshape or a blade-like shape are shown in FIG. 4.

The term “microcrystalline pellets” refers to a plurality ofmicrocrystals. Microcrystalline pellets are shown in FIG. 5 (viewedunder a microscopic).

The term “microcrystalline suspension” refers to a plurality ofmicrocrystals dispersed or suspended in an aqueous solution. In oneembodiment, the term “microcrystalline suspension” refers tomicrocrystalline pellets that are dispersed or suspended in an aqueoussolution.

The term “aqueous solution” is a solution in which the solvent is water.The aqueous solutions described herein may further comprisepharmaceutically acceptable excipients, as described herein.

The microcrystals are prepared by dissolving one or more Y receptoragonist peptides in an aqueous solution having a pH from about 3 toabout 5 to produce a first aqueous solution having the Y receptoragonist dissolved therein. The pH of the solution may be adjusted to apH from about 3 to about 5 using any buffer known in the art. Exemplarybuffers include phosphate buffers, acetate buffers, citrate buffers,glutamate buffers, and the like. In one embodiment, the buffer is aphosphate buffer. In one embodiment, the pH of the first aqueoussolution is from about 3.5 to about 4.5.

In one embodiment, the aqueous solution further comprises a dissolutionagent to facilitate or enhance dissolution of the Y receptor agonist.Any dissolution agent known in the art may be used. In one embodiment,the dissolution agent is a polyol. Exemplary polyols include glycerol,mannitol, sorbitol, sucrose, glycol, ethylene glycol, pentaerythritol,and the like. In one embodiment, the polyol is glycerol. The amount ofdissolution agent used will vary with the particular requirements of anyY receptor agonist.

After the Y receptor agonist is dissolved in the first aqueous solution,then the pH of the first aqueous solution is adjusted upward to a pH ofabout 4 to about 7.5 to produce a second aqueous solution having the Yreceptor agonist dissolved therein. The pH can be adjusted upward usingany buffer known in the art. Exemplary buffers include phosphatebuffers, Tris buffers, citrate buffers, glutamate buffers, and the like.In one embodiment, the buffer is a phosphate buffer. In one embodiment,the pH of the second aqueous solution is from about 4 to about 5.5. Inone embodiment, the pH of the second aqueous solution is from about 5.5to about 7.5.

Thereafter, microcrystals are formed by stirring the second aqueoussolution at a temperature of about 1° C. to about 10° C. for at leastone hour. The microcrystals begin to form during this process, until aplurality of microcrystals develop (substantially maintaining theircrystal structure) to form a microcrystalline suspension. In oneembodiment, the second aqueous solution is stirred at a temperature fromabout 1° C. to about 8° C. In one embodiment, the second aqueoussolution is stirred at a temperature from about 3° C. to about 6° C. Inone embodiment, the second aqueous solution is stirred for at least 12hours. In one embodiment, the second aqueous solution is stirred for atleast one day. In one embodiment, the second aqueous solution is stirredfrom about one day to about one month. In one embodiment, the secondaqueous solution is stirred from about one day to about ten days.

The microcrystalline suspension can be dried to produce microcrystallinepellets.

The final concentration of peptide in the microcrystalline suspensionmay be from 0.2 mg/mL to 10 mg/mL. The final concentration of peptide inthe microcrystalline may be determined by the pH of the second aqueoussolution. Generally, when the pH of the second aqueous solution is from4 to 5.5, the concentration of peptide in the microcrystallinesuspension is from 5 mg/mL to 10 mg/mL. Generally, when the pH of thesecond aqueous solution is from 5.5 to 7.5, the concentration of peptidein the microcrystalline suspension is from 0.2 mg/mL to 5 mg/mL.

As described in the Examples below, the individual microcrystals havestructures exemplified in FIG. 1 (viewed under a microscope) and FIG. 2(exhibiting birefringence when viewed under a polarized lightmicroscope). Upon drying the microcrystalline suspension, the resultingmicrocrystalline pellets (e.g., plurality or group of microcrystals)have the structures exemplified in FIG. 3.

Pharmaceutical compositions can be prepared using the microcrystallinesuspension and/or the microcrystalline pellets. Other pharmaceuticallyacceptable excipients known in the art and described herein may be addedto the pharmaceutical compositions to exhibit the desired pharmaceuticalproperties.

In another embodiment, the disclosure provides microcrystals of Yreceptor agonists coated with polyamino acids. The polyamino acids maybe any known in the art. The polyamino acids may be in the form ofpoly-L-amino acids. Exemplary poly-amino acids include poly-glutamicacid (poly-L-glutamic acid), poly-lysine (poly-L-lysine), poly-arginine(poly-L-arginine), poly-aspartic acid (poly-L-aspartic acid),poly-ornithine (poly-L-ornithine), and the like. In one embodiment, thepoly-amino acid is poly-L-glutamic acid. In one embodiment, themicrocrystals are coated with two different polyamino acids. Themicrocrystals can be partially coated, substantially coated, orcompletely coated with the polyamino acids, depending on the amount ofpolyamino acid used in the preparation. Similarly, the polyamino acidcoating can be any thickness, depending on the amount of polyamino acidused in the preparation. Following the methods described herein, theskilled artisan can vary and optimize the amount of polyamino acid usedin the process for coating the microcrystals.

The microcrystals are prepared by dissolving one or more Y receptoragonist peptides in an aqueous solution having a pH from about 3 toabout 5 to produce a first aqueous solution having the Y receptoragonist dissolved therein. The pH of the solution may be adjusted usingany buffer known in the art. Exemplary buffers include phosphatebuffers, acetate buffers, citrate buffers, glutamate buffers, and thelike. In one embodiment, the buffer is a phosphate buffer. In oneembodiment, the pH of the first aqueous solution is from about 3.5 toabout 4.5. In one embodiment, the aqueous solution further comprises adissolution agent to facilitate or enhance dissolution of the Y receptoragonist. Any dissolution agent known in the art may be used. In oneembodiment, the dissolution agent is a polyol. Exemplary polyols includeglycerol, mannitol, sorbitol, sucrose, glycol, ethylene glycol,pentaerythritol, and the like. In one embodiment, the polyol isglycerol.

After the Y receptor agonist is dissolved in the first aqueous solution,then the pH of the first aqueous solution is adjusted upward to a pH ofabout 4 to about 7.5 to produce a second aqueous solution. The pH can beadjusted upward using any buffer known in the art. Exemplary buffersinclude phosphate buffers, Tris buffers, citrate buffers, glutamatebuffers, and the like. In one embodiment, the buffer is a phosphatebuffer. In one embodiment, the pH of the second aqueous solution is fromabout 4 to about 5.5. In one embodiment, the pH of the second aqueoussolution is from about 5.5 to about 7.5.

Thereafter, microcrystals are formed by stirring the second aqueoussolution at a temperature of about 1° C. to about 10° C. for at leastone hour. The microcrystals begin to form during this process, until aplurality of microcrystals develop (substantially maintaining theircrystal structure) to form a microcrystalline suspension. In oneembodiment, the second aqueous solution is stirred at a temperature fromabout 1° C. to about 8° C. In one embodiment, the second aqueoussolution is stirred at a temperature from about 3° C. to about 6° C. Inone embodiment, the second aqueous solution is stirred for at least 12hours. In one embodiment, the second aqueous solution is stirred for atleast one day. In one embodiment, the second aqueous solution is stirredfrom about one day to about one month. In one embodiment, the secondaqueous solution is stirred from about one day to about ten days.

During or after formation of the microcrystalline suspension, one ormore polyamino acids are added to the microcrystalline suspension at thepH of the microcrystalline suspension and the microcrystallinesuspension and the polyamino acids are stirred at a temperature of about1° C. to about 10° C. for at least one hour. The polyamino acids may bein the form of an aqueous solution at a pH substantially the same as thepH of the microcrystalline suspension. In one embodiment, the polyaminoacids are added after formation of the microcrystalline suspension. Inone embodiment, the microcrystalline suspension and the polyamino acidsare stirred at a temperature from about 1° C. to about 8° C. In oneembodiment, the microcrystalline suspension and the polyamino acids arestirred at a temperature from about 3° C. to about 6° C. In oneembodiment, the microcrystalline suspension and the polyamino acids arestirred for at least 12 hours. In one embodiment, the microcrystallinesuspension and the polyamino acids are stirred for at least one day. Inone embodiment, the microcrystalline suspension and the polyamino acidsare stirred from about one day to about one month. In one embodiment,the microcrystalline suspension and the polyamino acids are stirred fromabout one day to about ten days. This results in a microcrystallinesuspension where the microcrystals are coated with the polyamino acid.The microcrystalline suspension can then be dried to producemicrocrystalline pellets where the microcrystals are coated with thepolyamino acid.

The final concentration of peptide in the microcrystalline suspensionmay be from 0.2 mg/mL to 10 mg/mL. The final concentration of peptide inthe microcrystalline may be determined by the pH of the second aqueoussolution. Generally, when the pH of the second aqueous solution is from4 to 5.5, the concentration of peptide in the microcrystallinesuspension is from 5 mg/mL to 10 mg/mL. Generally, when the pH of thesecond aqueous solution is from 5.5 to 7.5, the concentration of peptidein the microcrystalline suspension is from 0.2 mg/mL to 5 mg/mL.

In the methods described herein, the skilled artisan will be able to usean amount of polyamino acid to achieve a weight ratio ofpeptide:polyamino acid in the range of 1:1 to 10:1. In one embodiment,the weight ratio of peptide:polyamino acid may be in the range of 2:1 to6:1. In one embodiment, the weight ratio of peptide:polyamino acid maybe in the range of 4:1 to 6:1.

Pharmaceutical compositions can be prepared using the polyaminoacid-coated microcrystalline suspension and/or the polyamino acid-coatedmicrocrystalline pellets. Other pharmaceutically acceptable excipientsmay be added to the pharmaceutical compositions to exhibit the desiredpharmaceutical properties. In one embodiment, the pharmaceuticallyacceptable excipient is a preservative, such as meta-cresol.

The disclosure provides microcrystals complexed with one or more metals.The metals are generally in the form of salts. The metals are preferablyalkaline earth metals, transition metals or a combination thereof.Exemplary transition metals include zinc, iron, nickel, copper, and thelike. Exemplary alkaline earth metals include calcium, magnesium, andthe like. In one embodiment, the microcrystals are complexed with zinc,magnesium, calcium, or a combination of two or more thereof. In oneembodiment, the microcrystals are complexed with zinc.

The microcrystals are prepared by dissolving one or more Y receptoragonist peptides in an aqueous solution having a pH from about 3 toabout 5 to produce a first aqueous solution having the Y receptoragonist dissolved therein. The pH of the solution may be adjusted usingany buffer known in the art. Exemplary buffers include phosphatebuffers, acetate buffers, citrate buffers, glutamate buffers, and thelike. In one embodiment, the buffer is a phosphate buffer. In oneembodiment, the pH of the first aqueous solution is from about 3.5 toabout 4.5. In one embodiment, the aqueous solution further comprises adissolution agent to facilitate or enhance dissolution of the Y receptoragonist. Any dissolution agent known in the art may be used. In oneembodiment, the dissolution agent is a polyol. Exemplary polyols includeglycerol, mannitol, sorbitol, sucrose, glycol, ethylene glycol,pentaerythritol, and the like. In one embodiment, the polyol isglycerol.

Thereafter, one or more metals, preferably in the form of the metalsalt, are added to the first aqueous solution. Then the pH of the firstaqueous solution is adjusted upward to a pH of about 4 to about 7.5 toproduce a second aqueous solution. The pH can be adjusted using anybuffer known in the art. Exemplary buffers include phosphate buffers,Tris buffers, citrate buffers, glutamate buffers, and the like. In oneembodiment, the buffer is a phosphate buffer. In one embodiment, the pHof the second aqueous solution is from about 4 to about 5.5. In oneembodiment, the pH of the second aqueous solution is from about 5.5 toabout 7.5.

Thereafter, microcrystals complexed with the metals are formed bystirring the second aqueous solution at a temperature of about 1° C. toabout 10° C. for at least one hour. The metal-complexed microcrystalsbegin to form during this process, until a plurality of metal-complexedmicrocrystals develop (substantially maintaining their crystalstructure) to form a metal-complexed microcrystalline suspension. In oneembodiment, the second aqueous solution is stirred at a temperature fromabout 1° C. to about 8° C. In one embodiment, the second aqueoussolution is stirred at a temperature from about 3° C. to about 6° C. Inone embodiment, the second aqueous solution is stirred for at least 12hours. In one embodiment, the second aqueous solution is stirred for atleast one day. In one embodiment, the second aqueous solution is stirredfrom about one day to about one month. In one embodiment, the secondaqueous solution is stirred from about one day to about ten days.

The final concentration of peptide in the microcrystalline suspensionmay be from 0.2 mg/mL to 10 mg/mL. The final concentration of peptide inthe microcrystalline may be determined by the pH of the second aqueoussolution. Generally, when the pH of the second aqueous solution is from4 to 5.5, the concentration of peptide in the microcrystallinesuspension is from 5 mg/mL to 10 mg/mL. Generally, when the pH of thesecond aqueous solution is from 5.5 to 7.5, the concentration of peptidein the microcrystalline suspension is from 0.2 mg/mL to 5 mg/mL.

Based on the methods described herein, the skilled artisan will be ableto use an amount of metal to achieve a weight ratio of peptide:metal of1:1 to 10:1. In one embodiment, the weight ratio of peptide:metal is 1:1to 5:1. In one embodiment, the weight ratio of peptide:metal is 1:1 to4:1. In one embodiment, the weight ratio of peptide:metal is 1:1 to 3:1.In one embodiment, the weight ratio of peptide:metal is 1:1 to 2:1. Inone embodiment, the weight ratio of peptide:metal is 3:1. In oneembodiment, the weight ratio of peptide:metal is 2:1. In one embodiment,the weight ratio of peptide:metal is 1:1.

Pharmaceutical compositions can be prepared using the metal-complexedmicrocrystalline suspension and/or the metal-complexed microcrystallinepellets. Other pharmaceutically acceptable excipients may be added tothe pharmaceutical compositions to exhibit the desired pharmaceuticalproperties. In one embodiment, the pharmaceutically acceptable excipientis a preservative, such as meta-cresol.

The disclosure provides microcrystals complexed with one or more metalsand coated with polyamino acids. The metals are generally in the form ofsalts. The metals are preferably alkaline earth metals, transitionmetals or a combination thereof. Exemplary transition metals includezinc, iron, nickel, copper, and the like. Exemplary alkaline earthmetals include calcium, magnesium, and the like. In one embodiment, themicrocrystals are complexed with zinc, magnesium, calcium, or acombination of two or more thereof. In one embodiment, the microcrystalsare complexed with zinc. The polyamino acids may be any known in theart. The polyamino acids may be in the form of poly-L-amino acids.Exemplary poly-amino acids include poly-glutamic acid (poly-L-glutamicacid), poly-lysine (poly-L-lysine), poly-arginine (poly-L-arginine),poly-aspartic acid (poly-L-aspartic acid), poly-ornithine(poly-L-ornithine), and the like. In one embodiment, the poly-amino acidis poly-L-glutamic acid. In one embodiment, the microcrystals are coatedwith two different polyamino acids. The microcrystals can be partiallycoated, substantially coated, or completely coated with the polyaminoacids, depending on the amount of polyamino acid used in thepreparation. Similarly, the polyamino acid coating can be any thickness,depending on the amount of polyamino acid used in the preparation.Following the methods described herein, the skilled artisan can vary andoptimize the amount of polyamino acid used in the process for coatingthe microcrystals.

The microcrystals are prepared by dissolving one or more Y receptoragonist peptides in an aqueous solution having a pH from about 3 toabout 5 to produce a first aqueous solution having the Y receptoragonist dissolved therein. The pH of the solution may be adjusted usingany buffer known in the art. Exemplary buffers include phosphatebuffers, acetate buffers, citrate buffers, glutamate buffers, and thelike. In one embodiment, the buffer is a phosphate buffer. In oneembodiment, the pH of the first aqueous solution is from about 3.5 toabout 4.5. In one embodiment, the aqueous solution further comprises adissolution agent to facilitate or enhance dissolution of the Y receptoragonist. Any dissolution agent known in the art may be used. In oneembodiment, the dissolution agent is a polyol. Exemplary polyols includeglycerol, mannitol, sorbitol, sucrose, glycol, ethylene glycol,pentaerythritol, and the like. In one embodiment, the polyol isglycerol.

Thereafter, one or more metals, preferably in the form of the metalsalt, are added to the first aqueous solution. Then the pH of the firstaqueous solution is adjusted upward to a pH of about 4 to about 7.5 toproduce a second aqueous solution. The pH can be adjusted using anybuffer known in the art. Exemplary buffers include phosphate buffers,Tris buffers, citrate buffers, glutamate buffers, and the like. In oneembodiment, the buffer is a phosphate buffer. In one embodiment, the pHof the second aqueous solution is from about 4 to about 5.5. In oneembodiment, the pH of the second aqueous solution is from about 5.5 toabout 7.5.

Thereafter, microcrystals complexed with the metals are formed bystirring the second aqueous solution at a temperature of about 1° C. toabout 10° C. for at least one hour. The metal-complexed microcrystalsbegin to form during this process, until a plurality of metal-complexedmicrocrystals develop (substantially maintaining their crystalstructure) to form a metal-complexed microcrystalline suspension. In oneembodiment, the second aqueous solution is stirred at a temperature fromabout 1° C. to about 8° C. In one embodiment, the second aqueoussolution is stirred at a temperature from about 3° C. to about 6° C. Inone embodiment, the second aqueous solution is stirred for at least 12hours. In one embodiment, the second aqueous solution is stirred for atleast one day. In one embodiment, the second aqueous solution is stirredfrom about one day to about one month. In one embodiment, the secondaqueous solution is stirred from about one day to about ten days.

During or after formation of the metal-complexed microcrystallinesuspension, one or more polyamino acids are added to the metal-complexedmicrocrystalline suspension at the pH of the metal-complexedmicrocrystalline suspension and the metal-complexed microcrystallinesuspension and the polyamino acids are stirred at a temperature of about1° C. to about 10° C. for at least one hour. The polyamino acids may bein the form of an aqueous solution at a pH substantially the same as thepH of the metal-complexed microcrystalline suspension. In oneembodiment, the polyamino acids are added after formation of themetal-complexed microcrystalline suspension. In one embodiment, themetal-complexed microcrystalline suspension and the polyamino acids arestirred at a temperature from about 1° C. to about 8° C. In oneembodiment, the metal-complexed microcrystalline suspension and thepolyamino acids are stirred at a temperature from about 3° C. to about6° C. In one embodiment, the metal-complexed microcrystalline suspensionand the polyamino acids are stirred for at least 12 hours. In oneembodiment, the metal-complexed microcrystalline suspension and thepolyamino acids are stirred for at least one day. In one embodiment, themetal-complexed microcrystalline suspension and the polyamino acids arestirred from about one day to about one month. In one embodiment, themetal-complexed microcrystalline suspension and the polyamino acids arestirred from about one day to about ten days. This results in ametal-complexed microcrystalline suspension where the metal-complexedmicrocrystals are coated with the polyamino acid. The polyaminoacid-coated, metal-complexed microcrystalline suspension can then bedried to produce microcrystalline pellets where the microcrystals arecomplexed with metals and coated with the polyamino acid.

The final concentration of peptide in the microcrystalline suspensionmay be from 0.2 mg/mL to 10 mg/mL. The final concentration of peptide inthe microcrystalline may be determined by the pH of the second aqueoussolution. Generally, when the pH of the second aqueous solution is from4 to 5.5, the concentration of peptide in the microcrystallinesuspension is from 5 mg/mL to 10 mg/mL. Generally, when the pH of thesecond aqueous solution is from 5.5 to 7.5, the concentration of peptidein the microcrystalline suspension is from 0.2 mg/mL to 5 mg/mL. In themethods described herein, the skilled artisan will be able to use anamount of polyamino acid to achieve a weight ratio of peptide:polyaminoacid in the range of 1:1 to 10:1. In one embodiment, the weight ratio ofpeptide:polyamino acid may be in the range of 2:1 to 6:1. In oneembodiment, the weight ratio of peptide:polyamino acid may be in therange of 4:1 to 6:1. Based on the methods described herein, the skilledartisan will be able to use an amount of metal to achieve a weight ratioof peptide:metal of 1:1 to 10:1. In one embodiment, the weight ratio ofpeptide:metal is 1:1 to 5:1. In one embodiment, the weight ratio ofpeptide:metal is 1:1 to 4:1. In one embodiment, the weight ratio ofpeptide:metal is 1:1 to 3:1. In one embodiment, the weight ratio ofpeptide:metal is 1:1 to 2:1. In one embodiment, the weight ratio ofpeptide:metal is 3:1. In one embodiment, the weight ratio ofpeptide:metal is 2:1. In one embodiment, the weight ratio ofpeptide:metal is 1:1.

Pharmaceutical compositions can be prepared using the polyaminoacid-coated, metal-complexed microcrystalline suspension and/or thepolyamino acid-coated, metal-complexed microcrystalline pellets. Otherpharmaceutically acceptable excipients may be added to thepharmaceutical compositions to exhibit the desired pharmaceuticalproperties. In one embodiment, the pharmaceutically acceptable excipientis a preservative, such as meta-cresol.

Any Y receptor agonist peptide known in the art may be used to form themicrocrystals described herein. Exemplary Y receptor agonist peptidesare described in, e.g., U.S. Pat. No. 7,723,471; US Publication No.2006/0094653; US Publication No. 2010/0099619; PCT Publication No. WO2009/138511; PCT Publication No. WO 2011/033068; or PCT Publication No.WO 2011/058165, the disclosures of which are incorporated by referenceherein.

In one embodiment, the Y receptor agonist peptide is PYY(3-36) (SEQ IDNO:3). In one embodiment, the Y receptor agonist is a PYY analog.

“PYY analog” refers to a peptide that has at least 70%, at least 80%, atleast 85%, at least 90%o, at least 94%, or at least 97% sequenceidentity to PYY(3-36) over the entire length of PYY(3-36). In oneembodiment, the PYY analog has at least 80% sequence identity toPYY(3-36) over the entire length of PYY(3-36). In one embodiment, thePYY analog has at least 85% sequence identity to PYY(3-36) over theentire length of PYY(3-36). In one embodiment, the PYY analog has atleast 90% sequence identity to PYY(3-36) over the entire length ofPYY(3-36). In one embodiment, the PYY analog has at least 94% sequenceidentity to PYY(3-36) over the entire length of PYY(3-36). In oneembodiment, the PYY analog has at least 97% sequence identity toPYY(3-36) over the entire length of PYY(3-36).

In one embodiment, the PYY analog may be a chimeric peptide. In thisemobodiment, PYY(3-36) (SEQ ID NO:3) may comprise amino acid sequencesfrom the pancreatic polypeptide (“PP”) (SEQ ID NO: 1) and/orneuropeptide Y (SEQ ID NO:4) in order to vary the properties of thepeptide.

In other embodiments, the PYY analogs may be from 30 to 36 amino acidsin length; preferably from 32 to 34 amino acids in length. The skilledartisan will appreciate that PYY analogs may be prepared by the additionof amino acids to the amino acid sequence of PYY(3-36) (e.g., at theC-terminus, N-terminus, within the peptide, or a combination thereof);the deletion of amino acids from the amino acid sequence of PYY(3-36)(e.g., at the C-terminus, N-terminus, within the peptide, or acombination thereof); substitutions of any of the amino acids fordifferent amino acids in the amino acid sequence in PYY(3-6); or acombination of two or more of additions, deletions, and substitutions.As used herein, the term “substitution” includes modifications made toan amino acid (e.g., an amino acid is substituted with a modifiedversion of the original amino acid). Typical modifications to aminoacids include amides, carbohydrates, alkyl groups, acyl groups, fattyacyl groups, esters, and the like.

Exemplary Y receptor agonists are set forth in the Sequence Listingsubmitted herewith as part of the specification and incorporated byreference herein. Such Exemplary Y receptor agonists include SEQ ID NOs:1-483. These Y receptor agonist peptides are also described in U.S. Pat.No. 7,723,471.

In one embodiment the PYY analog has the amino acid sequence of SEQ IDNO:483:

Xaa₁ Lys Pro Glu Xaa₅ Pro Gly Glu Asp Ala Ser Pro Xaa₁₃ Glu Glu Leu AlaArg

Tyr Tyr Xaa₂₁ Xaa₂₂ Leu Arg Xaa₂₅ Tyr Ile Asn Leu Ile Thr Arg Gin ArgXaa₃₅, wherein Xaa₁ is Ile, isocaproyl-Ile, Pro, or isocaproyl-Pro; Xaa₅is His or Ala; Xaa₁₃ is Ala or absent; Xaa₂₁ is Ala or Ser; Xaa₂₂ is Seror Ala; Xaa₂₅ is Ala or His; and Xaa₃₅ is Tyr-OH or Tyr-NH₂.

In one embodiment, the peptide of SEQ ID NO:483 is SEQ ID NO:438: ProLys Pro Glu His Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Ala Arg Tyr TyrAla Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gin Arg Tyr (SEQ IDNO:438).

In one embodiment, the peptide of SEQ ID NO:483 is SEQ ID NO:281:isocaproyl-Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu LeuAla Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gin ArgTyr (SEQ ID NO:281).

In one embodiment, the peptide of SEQ ID NO:483 is SEQ ID NO:439: ProLys Pro Glu His Pro Gly Glu Asp Ala Ser Ala Glu Glu Leu Ala Arg Tyr TyrAla Ser Leu Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gin Arg Tyr (SEQ IDNO:439).

In one embodiment, the Y Receptor agonist comprises the amino acidsequence of SEQ ID NO:30. In one embodiment, the Y Receptor agonistcomprises the amino acid sequence of SEQ ID NO:88. In one embodiment,the Y Receptor agonist comprises the amino acid sequence of SEQ IDNO:348. In one embodiment, the Y Receptor agonist comprises the aminoacid sequence of SEQ ID NO:349. In one embodiment, the Y Receptoragonist comprises the amino acid sequence of SEQ ID NO:350. In oneembodiment, the Y Receptor agonist comprises the amino acid sequence ofSEQ ID NO:481. In one embodiment, the Y Receptor agonist comprises theamino acid sequence of SEQ ID NO:482.

In one embodiment, the Y receptor agonist is a PYY analog that has atleast 70% sequence identity to PYY(3-36) over the entire length ofPYY(3-36). In one embodiment, the Y receptor agonist is a PYY analogthat has at least 80% sequence identity to PYY(3-36) over the entirelength of PYY(3-36). In one embodiment, the Y receptor agonist is a PYYanalog that has at least 85% sequence identity to PYY(3-36) over theentire length of PYY(3-36). In one embodiment, the Y receptor agonist isa PYY analog that has at least 90% sequence identity to PYY(3-36) overthe entire length of PYY(3-36). In one embodiment, the Y receptoragonist is a PYY analog that has at least 94% sequence identity toPYY(3-36) over the entire length of PYY(3-36). In one embodiment, the Yreceptor agonist is a PYY analog that has at least 97% sequence identityto PYY(3-36) over the entire length of PYY(3-36).

In one embodiment, the Y receptor agonist is a compound described in PCTPublication No. WO 2009/138511. In one embodiment, the Y receptoragonist is a compound described in PCT Publication No. WO 2011/033068.In one embodiment, the Y receptor agonist is a compound described in WO2011/058165

The Y receptor agonist peptides described herein may be formulated aspharmaceutically acceptable salts (e.g., acid addition salts) and/orcomplexes thereof. Pharmaceutically acceptable salts include acidaddition salts such as those containing sulfate, hydrochloride,phosphate, sulfamate, acetate, citrate, lactate, tartrate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts canbe obtained from acids such as hydrochloric acid, sulfuric acid,phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid,tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,and quinic acid. Such salts may be prepared by, for example, reactingthe free acid or base forms of the product with one or more equivalentsof the appropriate base or acid in a solvent or medium in which the saltis insoluble, or in a solvent such as water which is then removed invacuo or by freeze-drying or by exchanging the ions of an existing saltfor another ion on a suitable ion exchange resin.

The compositions and pharmaceutical compositions described herein may beprovided in any form suitable for administration to a patient. In oneembodiment, the pharmaceutical compositions are in a form suitable fororal administration. In one embodiment, the pharmaceutical compositionsare in a form suitable for parenteral administration. Exemplary forms ofparenteral administration include infusion, intravenous, intramuscular,subcutaneous, and the like. In one embodiment, the compositions andpharmaceutical formulations are provided in a form suitable forsubcutaneous injection. The compositions and pharmaceutical compositionsmay be formulated with known pharmaceutically acceptable excipients,e.g., Remington's Pharmaceutical Sciences by E. W. Martin, including anaqueous solution.

Exemplary pharmaceutically acceptable excipients include buffers,isotonicity agents, preservatives, emulsifying agents, surfactants,chelating-agents, dissolution enhancing agents, and the like. Buffersinclude, for example, citrate buffers, acetate buffers, phosphatebuffers, glutamate buffers, and the like. Isotonicity agents include,for example, sodium chloride, dextrose, boric acid, sodium tartrate,propylene glycol, polyols, or other inorganic or organic solutes.Emulsifying agents include, for example, acacia powder, a non-ionicsurfactant, or an ionic surfactant). Preservatives include, for example,meta-cresol, sodium benzoate, potassium sorbate, phenol, methyl-paraben,ethyl-paraben, propyl-paraben, butyl-paraben, and the like. Otherpharmaceutically acceptable excipients include, for example, calciumcarbonate, calcium phosphate, sugars (e.g., lactose, glucose, sucrose),starch, cellulose derivatives, gelatin, vegetable oils, polyethyleneglycol, and the like. The skilled artisan will appreciate thatpharmaceutical compositions may contain one more excipients necessary toachieve the desired properties.

The pharmaceutical compositions described herein comprise the Y receptoragonist in therapeutically effective amounts. Typically, therapeuticallyeffective amounts will be in the range of about 0.1 μg to about 10mg/day; about 1 μg to about 5 mg/day; about 100 μg to about 1 mg/day; orabout 5 μg to about 500 μg/day. The exact dose to be administered may bedetermined by one of skill in the art and is dependent upon the potencyof the particular compound, as well as upon the age, weight andcondition of the individual.

It was unexpectedly discovered that the microcrystals of Y receptoragonists have extended pharmacokinetic (pK) profiles. Therefore, thepharmaceutical compositions containing the Y receptor agonists may beadministered to the patient once per day, once every other day, or onceper week.

The compounds, compositions, and pharmaceutical compositions describedherein may be used to treat any disease known in the art for whichtherapy with Y receptor agonists is appropriate. Exemplary diseases thatmay be treated using the compounds, compositions, and pharmaceuticalcompositions described herein include diabetes (e.g., type 1 diabetes,type 2 diabetes, gestational diabetes, pre-diabetes); obesity;overweight; fatty liver diseases (e.g., non-alcoholic steatohepatitis(NASH), nonalcoholic fatty liver disease (NAFLD)); gastrointestinaldiseases (e.g., ulcerative colitis, diarrhea, short bowel syndrome,small intestinal bacterial overgrowth, irritable bowel syndrome, Crohn'sdisease); gallbladder disease, hypertension, dyslipidemia,cardiovascular disease, myocardial infarction, atherosclerosis, eatingdisorders, insulin-resistance, and the like. In one embodiment, thecompounds, compositions, and pharmaceutical compositions describedherein are used to treat diabetes (e.g., type 1 diabetes, type 2diabetes, gestational diabetes, pre-diabetes) in a patient. The diabetesis preferably type 2 diabetes. In one embodiment, the compounds,compositions, and pharmaceutical compositions described herein are usedto treat obesity in a patient. In one embodiment, the compounds,compositions, and pharmaceutical compositions described herein are usedto treat overweight in a patient. In one embodiment, the compounds,compositions, and pharmaceutical compositions described herein are usedto treat fatty liver diseases (e.g., non-alcoholic steatohepatitis(NASH), nonalcoholic fatty liver disease (NAFLD) in a patient. In oneembodiment, the compounds, compositions, and pharmaceutical compositionsdescribed herein are used to treat gastrointestinal diseases (e.g.,ulcerative colitis, diarrhea, short bowel syndrome, irritable bowelsyndrome, Crohn's disease, gallbladder disease) in a patient.

The compounds, compositions, and pharmaceutical compositions describedherein may also be used to reduce body weight; reduce caloric intake;reduce food intake; slow gastric emptying; inhibit gastric acidsecretion; lower triglycerides; lower LDL cholesterol; inhibitpancreatic enzyme secretion; and alter body composition (e.g., decreasefat and increase lean muscle). In one embodiment, the compounds,compositions, and pharmaceutical compositions described herein are usedto reduce the body weight of a patient.

Each of the diseases and conditions described herein is known in the artand the art-recognized meaning and definition are intended to apply.Such diseases that can be treated with Y receptor agonists aredescribed, for example, in U.S. Pat. No. 7,723,471; US Publication No.2006/0094653; US Publication No. 2010/0099619; PCT Publication No. WO2009/138511; PCT Publication No. WO 2011/033068; or PCT Publication No.WO 2011/058165, the disclosures of which are incorporated by referenceherein.

EXAMPLES

The following examples are for purposes of illustration and are notintended to limit the scope of the claims.

Example 1

Y receptor agonists having SEQ ID NOs:438, 281 and 439 were synthesizedby known methods (e.g., U.S. Pat. No. 7,723,471) and purified by HPLC toa purity greater than 90%.

Example 2

SEQ ID NO:438 in the form of an acetate salt was dissolved in 30 mM, pH3 phosphate solution with 1.76% glycerol and 0.22% meta-cresol, andaseptically filtered through a 0.22 μm filter. The pH was then adjustedupward to pH 7.2±0.2 with 1 N sodium hydroxide solution. The totalweight of the solution was adjusted to the target weight using 30 mM, pH7.2 phosphate buffer with 1.76% glycerol and 0.22% meta-cresol. Amicrocrystalline suspension was obtained after stirring at a temperatureof about 5° C. for 7 days. The resulting concentration of peptide havingSEQ ID NO:438 was 2 mg/mL.

The morphology of the microcrystals was examined under microscope withor without polarized light. The crystals were oval shaped, as shown inFIG. 1. Under polarized light, the crystals exhibited birefringence, asshown in FIG. 2. The shape and surface morphology collected from themicrocrystalline suspension were confirmed by SEM. The particle size wasdetermined to be about 5-10 microns. The FTIR spectrum exhibited bothalpha-helical and beta-sheet secondary structural characteristics in thesolid crystals. Additionally, the FTIR spectrum of the solid crystalsshowed no significant difference compared to the original fluffy,amorphous powder prior to crystallization.

The skilled artisan will appreciate that microcrystalline suspensionwith a final concentration of SEQ ID NO:438 in the range of 0.2 mg/mL to10 mg/mL can be prepared by varying the amount of SEQ ID NO:438 acetateand phosphate buffer.

Example 3

A microcrystalline suspension comprising peptides having SEQ ID NO:281were prepared as described in Example 2, except that the pH was adjustedfrom pH 3 upward to a pH between 4 and 5.5. The concentration ofpeptides having SEQ ID NO:281 in the microcrystalline suspension was5-10 mg/ml. In this example, a microcrystalline suspension of thepeptide having a concentration in the range of 0.2 to 5 mg/mL could beformed at a higher pH range of 5.5 to 7.5 (rather than the pH of 4 to5.5 that was used).

Example 4

A microcrystalline suspension comprising peptides having SEQ ID NO:439was prepared as described in Example 2, except that the pH was adjustedfrom pH 3 upward to a pH from 6.5 to 7.4. The concentration of peptideshaving SEQ ID NO:439 in the buffered solution was 2 mg/ml.

Example 5

SEQ ID NO:438 acetate was dissolved in 30 mM, pH 3 phosphate solutionwith 1.76% glycerol and 0.22% meta-cresol, and aseptically filteredthrough a 0.22 μm filter. Zinc acetate solution was aseptically filteredand added to the solution. The pH was then adjusted upward to pH 7.2±0.2with 1 N sodium hydroxide solution. The total weight of the solution wasadjusted to the target weight using 30 mM, pH 7.2 phosphate buffer with1.76% glycerol and 0.22% meta-cresol. A zinc-complexed microcrystallinesuspension was obtained after constant stirring at a temperature ofabout 5° C. for 7 days. The final concentration of peptide was 2 mg/mL,and the weight ratio of peptide:zinc was 3:1.

The final concentration of the peptide having SEQ ID NO:438 in themicrocrystalline suspension having a range of 0.2-10 mg/mL can beprepared by varying the amount of SEQ ID NO:438 acetate, phosphatebuffer, and zinc acetate. Additionally, the weight ratio of peptide:zinccan be modified by varying the amount of zinc added to the solution.

Example 6

The microcrystalline suspensions of SEQ ID NO:438 without zinc (e.g.,Example 2) and with zinc (e.g., Example 5) were administered to maleSprague-Dawley rats (12 per formulation) by single subcutaneousinjection at 5 mg/kg dose. Vehicle (phosphate buffer) and a solutioncontaining SEQ ID NO:438 were used as controls. Blood samples werecollected at various time points from 0.25 to 72 hours post injection.Plasma concentrations of the peptide were determined by animmunoenzymetric assay (IEMA). PK parameters were calculated usingWinNonlin Professional software.

The results of the pK study are shown in FIG. 6. The results of the bodyweight study are shown in FIG. 7. With reference to FIGS. 6 and 7: Group1 was administered a phosphate buffer, pH 7.2, vehicle; Group 2 wasadministered a solution containing the peptide of SEQ ID NO:438; Group 3was administered a microcrystalline suspension containing the peptide ofSEQ ID NO:438; Group 4 was administered a zinc-complexedmicrocrystalline suspension of the peptide of SEQ ID NO:438, where theratio of peptide:zinc was 3:1; and Group 5 was administered azinc-complexed microcrystalline suspension of the peptide of SEQ IDNO:438, where the ratio of peptide:zinc was 1:1.

FIGS. 6 and 7 demonstrate that the results of rat PK and efficacystudies using microcrystalline suspensions of SEQ ID NO: 438 showedsustained release of the microcrystalline suspensions compared tovehicle and the solution containing SEQ ID NO:438. The mean plasmaconcentration of the peptide dropped to 2000 pg/mL at 8 hours postinjection for the control solution, while it took 72 hours to drop tothat same level for the microcrystalline suspensions. The rats thatreceived the microcrystalline suspensions showed approximately 10% morebody weight loss than the controls (vehicle and peptide solution) bysingle subcutaneous injection. The pK profile and weight loss efficacyof the microcrystalline suspension was not statistically significantlydifferent from that of the zinc-complexed microcrystalline suspension.

Example 7

A poly-L-glutamic acid solution was prepared as follows. Poly-L-glutamicacid was dissolved in purified and de-ionized water. The pH was adjustedto pH 7.2 with IN sodium hydroxide solution.

SEQ ID NO:438 acetate was dissolved in 30 mM, pH 3 phosphate solutionwith 1.76% glycerol and 0.22% meta-cresol, and aseptically filteredthrough a 0.22 μm filter. The pH was then adjusted upward to pH 7.2±0.2with 1 N sodium hydroxide solution. The total weight of the solution wasadjusted to the target weight using 30 mM, pH 7.2 phosphate buffer with1.76%) glycerol and 0.22% meta-cresol. A microcrystalline suspension wasobtained after stirring at 5° C. for 7 days.

Thereafter, the poly-L-glutamic acid solution at pH 7.2 was added to themicrocrystalline suspension while stirring. Poly-L-glutamic acid-coatedmicrocrystalline suspension was formed after constant stirring at 5° C.for 4 days. The final concentration of peptide was 2 mg/mL and theweight ratio of peptide to poly-L-glutamic acid was 5:1.

Poly-L-glutamic acid-coated microcrystalline suspension with a finalconcentration of SEQ ID NO:438 in the range of 1-10 mg/mL can beprepared by varying the amount of SEQ ID NO:438 acetate, phosphatebuffer, and poly-L-glutamic acid.

Example 8

Animal studies were undertaken to compare the formulations describedherein for effects on food intake. The following formulations were used:(1) vehicle/placebo containing 30 mM phosphate buffer with 1.76%glycerol at pH 7.2; (ii) solution containing the peptide of SEQ IDNO:438; 4.9% mannitol, 0.07% Tween 80; and a 10 mM acetate buffer, at apH 4.3; (iii) the microcrystalline suspension of Example 2; (iv) thepolyamino acid-coated microcrystalline suspension of Example 7; and (v)a solution of a pegylated peptide, i.e., the peptide of SEQ ID NO:438modified with K¹¹ conjugated to a linear polyethylene glycol having amolecular weight of 20 kilodaltons. The formulations were injectedsubcutaneously (100 μg/kg) into overnight-fasted female NIH/Swiss mice.Food was introduced immediately after injection and the amount consumewas measured at 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240minutes, 300 minutes, 360 minutes, 24 hours, 30 hours, 48 hours, 56hours, and 72 hours. The results for cumulative food intake are shown inFIG. 8 (*p<0.05 vs. vehicle control; ANOVA, Dunnett's test). The resultsfor the change in food intake from baseline are shown in FIG. 9 (*p<0.05compared to placebo; ̂p<0.05 compared to solution containing the peptideof SEQ ID NO:438; 4.9% mannitol, 0.07% Tween 80; and a 10 mM acetatebuffer, at a pH 4.3).

1. A microcrystal comprising a Y receptor agonist peptide.
 2. Themicrocrystal of claim 1, wherein the Y receptor agonist peptidecomprises the amino acid sequence of any one of SEQ ID NOs: 1-483. 3-6.(canceled)
 7. The microcrystal of claim 1, wherein the Y receptoragonist peptide has at least 80% sequence identity to PYY(3-36). 8-12.(canceled)
 13. The microcrystal of claim 1, wherein the microcrystal hasa particle size of 1 micron to 15 microns.
 14. (canceled)
 15. Themicrocrystal of claim 1, wherein the microcrystal is birefringent whenviewed under a polarized light microscope.
 16. A pharmaceuticalcomposition comprising a plurality of microcrystals according toclaim
 1. 17. The pharmaceutical composition of claim 16 furthercomprising an aqueous solution.
 18. (canceled)
 19. The pharmaceuticalcomposition of claim 17, further comprising polyol.
 20. Thepharmaceutical composition of claim 19, wherein the polyol is glycerol,mannitol, sorbitol, sucrose, glycol, ethylene glycol, pantaerythritol,or a combination thereof. 21-31. (canceled)
 32. A microcrystalcomprising a Y receptor agonist peptide and a polyamino acid.
 33. Themicrocrystal of claim 32, wherein the Y receptor agonist peptidecomprises the amino acid sequence of airy one of SEQ ID NOs: 1-483.34-37. (canceled)
 39. The microcrystal of claim 32, wherein the Yreceptor agonist peptide has at least 80% sequence identity toFYY(3-36). 39-43. (canceled)
 44. The microcrystal of claim 32, whereinthe microcrystal has a particle size of 1 micron to 15 microns. 45.(canceled)
 46. The microcrystal of claim 32, wherein the microcrystal isbirefringent when viewed under a polarized light microscope.
 47. Themicrocrystal of claim 32, wherein the microcrystal is coated with thepolyamino acid.
 48. The microcrystal of claim 32, wherein the polyaminoacid is poly-glutamic acid, poly-lysine, poly-arginine, poly-asparticacid, poly-leucine, poly-ornithine, or a combination of two or morethereof. 49-57. (canceled)
 58. A microcrystal comprising a Y receptoragonist peptide and a metal.
 59. The microcrystal of claim 58, whereinthe Y receptor agonist peptide comprises the amino acid sequence of anyone of SEQ ID NOs: 1-483. 60-63. (canceled)
 64. The microcrystal ofclaim 58, wherein the Y receptor agonist peptide has at least 80%sequence identity to PYY(3-36). 65-69. (canceled)
 70. The microcrystalof claim 58, wherein the microcrystal has a particle size of 1 micron to15 microns.
 71. (canceled)
 72. The microcrystal of claim 58, wherein themicrocrystal is birefringent when viewed under a polarized lightmicroscope.
 73. The microcrystal of claim 58, wherein the metal is analkaline earth metal a transition metal or a combination thereof. 74.The microcrystal of claim 58, wherein the metal is sine, iron, nickel,copper, calcium, magnesium, or a combination thereof. 75-84. (canceled)85. A method for treating diabetes, overweight, obesity, fatty liverdisease, a gastrointestinal disease, hypertension, dyslipidemia, acardiovascular disease, a myocardial infarction, atherosclerosis, aneating disorder, or insulin-resistance in a patient in need thereofcomprising administering a therapeutically effective amount of apharmaceutical composition comprising the microcrystal of claim 1.86-99. (canceled)